Mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant form of focal and segmental glomerulosclerosis (FSGS). ACTN4-mediated FSGS is dominant, adult onset, and slowly progressive, suggesting subtle time and stress dependent alterations in podocyte function. The hypothesis guiding our studies is that dominant, gain-of-function mutations in the actin-binding domain of alpha-actinin-4 leads to an altered actin cytoskeleton, altered mechanical function, and a decreased ability to respond to stress. We will continue our efforts to understand this kidney disease, utilizing new biochemical, cellular, and animal tools that we have developed. We aim to: (1) Define the effect of mutant alpha-actinin-4 on the biomechanical properties of the actin cytoskeleton and on actin dynamics: We will determine if alpha-actinin-4 mutations act in a gain-of-function manner to alter the structure of the actin filament network and actin dynamics. We will determine the effect of alpha-actinin-4 mutations on actin dynamics and polymerization in vitro and in vivo, as well as the effect on the ultrastructure of the actin cytoskeleton. We will also examine if the actin and alpha-actinin-4-associated molecule synaptopodin alters the biochemical properties of mutant alpha-actinin-4. (2) Define the downstream effects of mutant alpha-actinin-4 biology on cellular processes: We will determine if ACTN4-mediated alterations in the cytoskeleton have detrimental cellular consequences. We will examine the biomechanical properties of mutant cells. We will examine if aggregation of alpha-actinin and actin caused by disease-associated mutations has toxic effects on proteasome function. We will also determine if the cytoskeletal changes lead to altered mitochondrial function, increased ROS production, and apoptosis. (3) Define the role of envirionmental and genetic factors on glomerular function in Actn4-mutant disease in vivo: We will stress mice to determine if heterozygosity for K255E in mice increases susceptibility to glomurular injury. We will treat mice with angiotensin converting enzyme inhibitors, to determine if reducing the mechanical forces on mutant podocytes alters disease progression. Because synaptopodin appears to modulate the alpha-actinin-4/F-actin interaction, we will examine this interaction genetically by generating and phenotyping synaptopodin-deficient/alpha-actinin-4 mutant mice. Public health relatedness: Kidney failure is an increasing cause of morbidity and mortality. FSGS is a form of kidney disease of increasing frequency. Our studies will help understand how defects in a particular human gene ACTN4 lead to kidney disease and, ultimately, may give new insights into treatment of FSGS.